1. Intermediates and Mechanism of Amyloid Formation[unreadable] [unreadable] Fluorescence energy transfer (FET) kinetics provide structural information because rates depend on the inverse sixth power of the distance between a fluorescent donor (D) and energy acceptor (A). A DA pair is characterized by a critical length (ro) that defines the range of distances (r) that can be probed (0.1ro < r < 1.5ro). DA partners incorporated into a single peptide will furnish site-specific conformational detail. In heterogeneous systems, such as unstructured and fibril-forming polypeptides, analysis of FET kinetics can provide DA distance distributions. Measurements of FET kinetics in dye-labeled amyloidogenic peptides will permit us to probe for transient intermediates generated during aggregation.[unreadable] [unreadable] In order to identify crucial regions for protein-protein interactions, as well as a putative nucleation site for aggregation, we have engineered six single Cys synuclein variants for introduction of fluorescent donor and acceptor molecules. These multiple sites will allow us to probe specific regions of the protein, the N-terminal (Cys7, Cys 26, Cys57), hydrophobic central (Cys66 and Cys88), and acidic C-terminal (Cys100) region. We have successfully derivatized the Cys7 protein with 5-(2-(acetylamino)ethyl)naphthalene-1-sulfonic acid fluorophore (Dns) and the Cys88 mutant with 5-methyl-fluorescein (mFl). Additional sites are provided by single Trp mutants at positions 4, 39, 94, and 125. With these three different fluorescent labels, we are able to investigate interprotein distances ranging from 10 to 70 angstroms (Trp-to-Dns, ro = 31 angstroms and Dns-to-mFl, ro = 46 angstroms). These intermolecular FET reactions will be used to identify and characterize soluble aggregates as functions of concentration, temperature, and additives, in particular, phospholipid vesicles and metal ions. Interprotein energy transfer with 1:10 mixtures of D- and A-containing mutants will provide direct evidence for oligomerization. In favorable cases, variations in FET kinetics as a function of protein concentration will be used to estimate dissociation constants for soluble aggregates. By varying the DA sites, interprotein contacts will be revealed.[unreadable] [unreadable] 2. Copper(II) Binding to alpha-Synuclein[unreadable] [unreadable] Although recent work points to a genetic component to Parkinsons disease (PD) involving the accumulation and deposit of a neuronal protein, alpha-synuclein, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-synuclein in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work.[unreadable] [unreadable] To probe for Cu(II)-protein binding, we tested three mutant proteins containing single W (F4W, F94W, Y39W) substitutions. Upon addition of Cu(II) (0.25-5 micromolar), the tryptophan fluorescence intensity decreases. Unexpectedly, the fluorophore at position 4 exhibits the most quenching in the presence of the metal ion (50% at 1.0 eq. Cu(II)). In contrast, W39, which is closest to H50, a ligand assigned by NMR, exhibits minimal quenching until more than one equivalent of Cu(II) has been added (protein =5 micromolar). Similarly, W94 does not respond to substoichiometric Cu(II) concentrations. Our data indicate that there are multiple Cu(II) binding sites on alpha-synuclein. Because W4 appears to be the most sensitive reporter, we aim to examine Cu(II) binding with mutant F4W in more depth, in order to probe the N-terminal region as well as to ensure that removal of native tyrosine residues does not affect metal-ion interactions with the protein. In addition, we have replaced H50 with a serine residue (F4W/H50S variant) to assess the role, if any, of imidazole ligation to Cu(II).[unreadable] [unreadable] These preliminary results clearly demonstrate that tryptophan fluorescence is a sensitive indicator of copper(II) binding to alpha-synuclein. Current work is focused on elucidating the role of Cu(II)-synuclein conjugates in promoting misfolding and aggregation.